Primaquine, an 8-aminoquinoline, with clindamycin is the most effective clinical alternative to standard therapy for patients with pneumonia caused by Pneumocystis, but the mechanism of action of 8-aminoquinolines against the organism and against others such as Plasmodium remains unknown. Therefore, using primaquine as the immobilized ligand we have used affinity chromatography to isolate proteins from Pneumocystis with high affinity for primaquine. Preliminary identification of one of these proteins suggests it is an intermediate filament protein, which may relate to the profound cytoplasmic disruption produced in Pneumocystis by 8-aminoquinolines. To establish the role of primaquine binding proteins in the mechanism of action of primaquine against Pneumocystis, we propose the following specific aims. Specific Aim 1: Isolate and identify additional primaquine binding proteins from Pneumocystis. 1a. Purify proteins from Pneumocystis using primaquine affinity chromatography. 1b. Sequence peptide fragments of the proteins using mass spectrometry. 1c. Use bioinformatics and mass spectrometry data to search for homologs. 1d. Clone: full-length gene product of putative primaquine-binding proteins. Specific Aim 2: Use existing protein sequence data to identify the coding sequences for Pneumocystis intermediate filament protein(s). 2a. Clone by degenerate PCR/RACE based on alignments, codon bias in the organism, and available Pneumocystis genome database information. 2b. Identify the full length transcript from Pneumocystis RNA. 2c. Express and purify full length recombinant proteins from Escherichia coli. Achieving a clear understanding of the mechanism of action of 8-aminoquinolines toward Pneumocystis will inform similar studies in the future with Plasmodium. Thus, this project could have significant impact upon therapy of both an opportunistic infection affecting thousands of patients and a world-wide disease that affects millions of persons each year. [unreadable] [unreadable]